Method of determining dihydropyrimidine dehydrogenase gene expression

ABSTRACT

The present invention relates to prognostic methods which are useful in medicine, particularly cancer chemotherapy. The object of the invention to provide a method for assessing Dihydropyrimidine dehydrogenase (DPD) expression levels in tissues and prognosticate the probable resistance of a patient&#39;s tumor to treatment with 5-FU based therapies by examination of the amount of DPD mRNA in a patient&#39;s tumor cells and comparing it to a predetermined threshold expression level. More specifically, the invention provides to oligonucleotide primer pairs DPD3A and DPD3B and methods comprising their use for detecting levels of Dihydropyrimidine dehydrogenase (DPD) mRNA.

FIELD OF THE INVENTION

The present invention relates to prognostic methods which are useful inmedicine, particularly cancer chemotherapy. The invention also relatesto assessment of gene expression of tumor cells of a patient. Morespecifically, the invention relates to oligonucleotides and methodscomprising their use for detecting levels of Dihydropyrimidinedehydrogenase (DPD) mRNA expression using RT-PCR.

BACKGROUND OF THE INVENTION

Cancer arises when a normal cell undergoes neoplastic transformation andbecomes a malignant cell. Transformed (malignant) cells escape normalphysiologic controls specifying cell phenotype and restraining cellproliferation. Transformed cells in an individual's body thusproliferate in the absence of these normal controls, thus forming atumor. When a tumor is found, the clinical objective is to destroymalignant cells selectively while mitigating any harm caused to normalcells in the individual undergoing treatment.

Chemotherapy is based on the use of drugs that are selectively toxic(cytotoxic) to cancer cells. Several general classes of chemotherapeuticdrugs have been developed, including drugs that interfere with nucleicacid synthesis, protein synthesis, and other vital metabolic processes.

5-Fluorouracil (5-FU) is a very widely used drug used for the treatmentof many different types of cancers, including major cancers such asthose of the GI tract and breast (Moertel, C. G. New Engl. J. Med.,330:1136-1142, 1994). 5-FU as a single agent was for more than 40 yearsthe standard first-line treatment for colorectal cancer, but it wassupplanted as “standard of care” by the combination of 5-FU and CPT-11(Saltz et al., Irinotecan Study Group. New England Journal of Medicine.343:905-14, 2000). Recently, the combination of 5-FU and oxaliplatin hasproduced high response rates in colorectal cancers (Raymond et al.,Semin. Oncol., 25:4-12, 1998). Thus, it is likely that 5-FU will be usedin cancer treatment for many years because it remains the centralcomponent of current chemotherapeutic regimens. In addition, singleagent 5-FU therapy continues to be used for patients in whom combinationtherapy with CPT-11 or oxaliplatin is likely to be excessively toxic.

5-FU is typical of most anti-cancer drugs in that only the minority ofpatients experience a favorable response to the therapy. Largerandomized clinical trials have shown the overall response rates oftumors to 5-FU as a single agent for patients with metastatic colorectalcancer to be in the 15-20% range (Moertel, C. G. New Engl. J. Med.,330:1136-1142, 1994). In combination with other chemotherapeuticsmentioned above, tumor response rates to 5-FU-based regimens have beenincreased to almost 40%. Nevertheless, the majority of treated patientsderive no tangible benefit from having received 5-FU based chemotherapy,and are subjected to significant risk, discomfort, and expense. Sincethere has been no reliable means of anticipating the responsiveness ofan individual's tumor prior to treatment, the standard clinical practicehas been to subject all patients to 5-FU-based treatments, fullyrecognizing that the majority will suffer an unsatisfactory outcome.

The mechanism of action and the metabolic pathway of 5-FU have beenintensively studied over the years to identify the most importantbiochemical determinants of the drug's anti-tumor activity. The ultimategoal was to improve the clinical efficacy of 5-FU by a) modulation ofits intracellular metabolism and biochemistry and b) by measuringresponse determinants in patients' tumors prior to therapy to predictwhich patients are most likely to respond (or not to respond) to thedrug. Two major determinants emerged from these studies: 1) the targetenzyme of 5-FU, thymidylate synthase (TS) and 2) the catabolic enzymedihydropyrimidine dehydrogenase (DPD).

The first studies in the area of tumor response prediction to 5-FU basedtherapy centered on the target enzyme TS in colorectal cancer. Leichmanet al (Leichman et al., J. Clin Oncol., 15:3223-3229, 1997) carried outa prospective clinical trial to correlate tumor response to 5-FU with TSgene expression as determined by RT-PCR in pre-treatment biopsies fromcolorectal cancers. This study showed: 1) a large 50-fold range of TSgene expression levels among these tumors, and 2) strikingly differentlevels of TS gene expression between responding and non-respondingtumors. The range of TS levels of the responding groups (0.5-4.1,relative to an internal control) was narrower than that of thenon-responding groups (1.6-23.0, relative to an internal control). Theinvestigators determined a resulting “non-response cutoff” thresholdlevel of TS expression above which there were only non-responders. Thus,patients with TS expression above this “non-response cutoff” thresholdcould be positively identified as non-responders prior to therapy. The“no response” classification included all therapeutic responses with<50% tumor shrinkage, progressing growth resulting in a >25% tumorincrease and non-progressing tumors with either <50% shrinkage, nochange or <25% increase. These tumors had the highest TS levels. Thus,high TS expression identifies especially resistant tumors. TS expressionlevels above a certain threshold identified a subset of tumors notresponding to 5-FU, whereas TS expression levels below this numberpredicted an appreciably higher response rate yet did not specificallyidentify responding tumors.

Subsequent studies investigated the usefulness of DPD expression levelsas a tumor response determinant to 5-FU treatment in conjunction with TSexpression levels. DPD is a catabolic enzyme which reduces the 5,6double bond of 5-FU, rendering it inactive as a cytotoxic agent.Previous studies have shown that DPD levels in normal tissues couldinfluence the bio-availability of 5-FU, thereby modulating itspharmacokinetics and anti-tumor activity (Harris et al., Cancer Res.,50: 197-201, 1990). Additionally, evidence has been presented that DPDlevels in tumors are associated with sensitivity to 5-FU (Etienne etal., J. Clin. Oncol., 13: 1663-1670, 1995; Beck et al., Eur. J. Cancer,30: 1517-1522, 1994). Salonga et al, (Clin Cancer Res., 6:1322-1327,2000) investigated gene expression of DPD as a tumor responsedeterminant for 5-FU/leucovorin treatment in a set of tumors in which TSexpression had already been determined. As with TS, the range of DPDexpression among the responding tumors was relatively narrow (0.6-2.5,4.2-fold; relative to an internal control) compared with that of thenon-responding tumors (0.2-16, 80-fold; relative to an internalcontrol). There were no responding tumors with a DPD expression greaterthan a threshold level of about 2.5. Furthermore, DPD and TS expressionlevels showed no correlation with one another, indicating that they areindependently regulated genes. Among the group of tumors having both TSand DPD expression levels below their respective “non-response cut-off”threshold levels, 92% responded to 5-FU/LV. Thus, responding tumorscould be identified on the basis of low expression levels of DPD and TS.

DPD is also an important marker for 5-FU toxicity. It was observed thatpatients with very low DPD levels (such as in DPD Deficiency Syndrome;i.e. thymine uraciluria) undergoing 5-FU based therapy suffered fromlife-threatening toxicity (Lyss et al., Cancer Invest., 11: 2390240,1993). Indeed, the importance of DPD levels in 5-FU therapy wasdramatically illustrated by the occurrence of 19 deaths in Japan from anunfavorable drug interaction between 5-FU and an anti-viral compoundSorivudine (Diasio et al., Br. J. Clin. Pharmacol. 46, 1-4, 1998) It wassubsequently discovered that a metabolite of Sorivudine is a potentinhibitor of DPD. This treatment resulted in DPD DeficiencySyndrome-like depressed levels of DPD which increased the toxicity of5-FU to the patients (Diasio et al., Br. J. Clin. Pharmacol. 46, 1-4,1998).

Thus, because of a) the widespread use of 5-FU protocols in cancertreatment, b) the important role of DPD expression in predicting tumorresponse to 5-FU and c) the sensitivity of individuals withDPD-Deficiency Syndrome to common 5-FU based treatments, it is clearthat accurate determination of DPD expression levels prior tochemotherapy will provide an important benefit to cancer patients.

Measuring DPD enzyme activity requires a significant amount of freshtissue that contains active enzyme. Unfortunately, most pre-treatmenttumor biopsies are available only as fixed paraffin embedded (FPE)tissues, particularly formalin-fixed parafin embedded tissues which donot contain active enzyme. Moreover, biopsies generally contain only avery small amount of heterogeneous tissue.

RT-PCR primer and probe sequences are available to analyze DPDexpression in frozen tissue or fresh tissue. However, those primers areunsuitable for the quantification of DPD mRNA from fixed tissue byRT-PCR. Heretofore, existing primers give no or erratic results. This isthought to be due to the a) inherently low levels of DPD RNA; b) verysmall amount of tissue embedded in the paraffin; and c) degradation ofRNA in the paraffin into short pieces of <100 bp. As a result, otherinvestigators have made a concerted yet unsuccessful efforts to obtainoligonucleotide primer sets allowing for such a quantification of DPDexpression in paraffinized tissue. Thus, there is a need for method ofquantifying DPD mRNA from fixed tissue in order to provide an earlyprognosis for proposed cancer therapies. Because it has been shown thatDPD enzyme activity and corresponding mRNA expression levels are wellcorrelated (Ishikawa et al., Clin. Cancer Res., 5:883-889, 1999; Johnsonet al., Analyt. Biochem. 278: 175-184, 2000), measuring DPD mRNAexpression in FPE specimens provides a way to assess the DPD expressionlevels status of patients without having to determine enzyme activity infresh tissues. Furthermore, FPE specimens are readily amenable tomicrodissection, so that DPD gene expression can be determined in tumortissue uncontaminated with stromal tissue.

Accordingly, it is the object of the invention to provide a method forassessing DPD levels in tissues and prognosticate the probableresistance of a patient's tumor to treatment with 5-FU based therapies,by examination of the amount of DPD mRNA in a patient's tumor cells andcomparing it to a predetermined threshold expression level.

SUMMARY OF THE INVENTION

In one aspect of the invention there is provided oligonucleotide primershaving the sequence of DPD3A-51F (SEQ ID NO: 1) or DPD3A-134R (SEQ IDNO:2), as well as oligionucleotide primers DPD3b-651F (SEQ ID NO: 7) andDPD3b-736R (SEQ ID NO: 8) and sequences substantially identical thereto.The invention also provides for oligonucleotide primers having asequence that hybridizes with DPD3A-51F (SEQ ID NO: 1), DPD3A-134R (SEQID NO: 2), DPD3b-651F (SEQ ID NO:7), DPD3b-736R (SEQ ID NO: 8) orcomplements thereof under stringent conditions.

Moreover, this invention relates to a method for determining achemotherapeutic regimen, comprising obtaining an mRNA sample from atumor specimen; determining DPD gene expression level in the specimen;comparing the determined DPD gene expression levels with a predeterminedthreshold level for that gene; and determining a chemotherapeuticregimen based on the results of the comparison of the determined geneexpression level with the predetermined threshold level.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing a comparison of four differentoligionucleotide primer pairs for their ability to amplify DPD mRNAderived from 10 different formalin-FPE tissue samples. Samples #1-5, and#8-10 are derived from colon tumor, #6 from bronchoalveolar tumor and #7from small bowel tumor biopsies. Oligonucleotide primer pairs DPD1(DPD-70F, (SEQ ID NO: 3) and DPD-201R, (SEQ ID NO: 4)), DPD2(DPD2p-1129F (SEQ ID NO: 5) and DPD2p-1208R (SEQ ID NO: 6)) are noteffective in measuring DPD mRNA levels in these samples. Oligonucleotideprimer pairs DPD3A (DPD3a-51F (SEQ ID NO: 1) and DPD3a-134R (SEQ ID NO:2)) and DPD3B (DPD3b-651F (SEQ ID NO: 7) and DPD3b-736R (SEQ ID NO: 8))are effective in ascertaining DPD levels in various samples.

FIG. 2 is a graph showing a comparison of DPD mRNA amplificationefficiency of the oligonucleotide primer pairs DPD3A (DPD3a-51F (SEQ IDNO: 1) and DPD3a-134R (SEQ ID NO: 2)) and DPD1 (DPD-70F (SEQ ID NO: 3)and DPD-201R (SEQ ID NO: 4)) in frozen tissue samples. The graphillustrates that not only is the oligonucleotide primer pair DPD3A(DPD3a-51F (SEQ ID NO: 1) and DPD3a-134R (SEQ ID NO: 2)) also effectivein measuring DPD expression levels in frozen tissue samples (as well asFPE derived samples) it is more efficient than the oligonucleotideprimer pair DPD1 (DPD-70F (SEQ ID NO: 3) and DPD-201R (SEQ ID NO: 4)).

DETAILED DESCRIPTION OF THE INVENTION

The present inventors disclose oligonucleotide primers andoligonucleotide primers substantially identical thereto that allowaccurate assessment of DPD expression in tissues. These oligonucleotideprimers, DPD3 a-51F (SEQ ID NO: 1) and DPD3a-134R (SEQ ID NO: 2), (alsoreferred to herein as the oligonucleotide primer pair DPD3A) andoligionucleotide primers DPD3b-651F (SEQ ID NO: 7) and DPD3b-736R (SEQID NO: 8), (also referred to herein as the oligonucleotide primer pairDPD3B) are particularly effective when used to measure DPD geneexpression in fixed paraffin embedded (FPE) tumor specimens.

“Substantially identical” in the nucleic acid context as used herein,means that the oligonucleotides hybridizes to a target under stringentconditions, and also that the nucleic acid segments, or theircomplementary strands, when compared, are the same when properlyaligned, with the appropriate nucleotide insertions and deletions, in atleast about 60% of the nucleotides, typically, at least about 70%, moretypically, at least about 80%, usually, at least about 90%, and moreusually, at least, about 95-98% of the nucleotides. Selectivehybridization exists when the hybridization is more selective than totallack of specificity. See, Kanehisa, Nucleic Acids Res., 12:203-213(1984).

This invention includes substantially identical oligonucleotides thathybridize under stringent conditions (as defined herein) to all or aportion of the oligonucleotide primer sequence of DPD3A-51F (SEQ IDNO:1), its complement, DPD3A-134R (SEQ ID NO: 2) or its complement.Furthermore, this invention also includes substantially identicaloligonucleotides that hybridize under stringent conditions (as definedherein) to all or portion of the oligonucleotide primer sequenceDPD3b-651F (SEQ ID NO: 7) its complement, DPD3b-736R (SEQ ID NO: 8), orits complement.

Under stringent hybridization conditions, only highly complementary,i.e., substantially similar nucleic acid sequences hybridize.Preferably, such conditions prevent hybridization of nucleic acidshaving 4 or more mismatches out of 20 contiguous nucleotides, morepreferably 2 or more mismatches out of 20 contiguous nucleotides, mostpreferably one or more mismatch out of 20 contiguous nucleotides.

The hybridizing portion of the nucleic acids is typically at least 10(e.g., 15) nucleotides in length. The hybridizing portion of thehybridizing nucleic acid is at least about 80%, preferably at leastabout 95%, or most preferably about at least 98%, identical to thesequence of a portion or all of oligonucleotide primer DPD3A-51F (SEQ IDNO: 1), its complement, DPD3A-134R (SEQ ID NO: 2) or its complement.Additionally, the hybridizing portion of the hybridizing nucleic acid isat least least about 80%, preferably at least about 95%, or mostpreferably about at least 98%, identical to the sequence of a portion orall of oligonucleotide primer DPD3b-651F (SEQ ID NO: 7), its complement,DPD3b-736R (SEQ ID NO: 8) or its complement.

Hybridization of the oligonucleotide primer to a nucleic acid sampleunder stringent conditions is defined below. Nucleic acid duplex orhybrid stability is expressed as a melting temperature (T_(m)), which isthe temperture at which the probe dissociates from the target DNA. Thismelting temperature is used to define the required stringencyconditions. If sequences are to be identified that are substantiallyidentical to the probe, rather than identical, then it is useful tofirst establish the lowest temperature at which only holmologoushybridization occurs with a particular concentration of salt (e.g. SSCor SSPE). Then assuming that 1% mismatching results in a 1° C. decreasein T_(m), the temperatre of the final wash in the hybridization reactionis reduced accordingly (for example, if sequences having >95% identitywith the probe are sought, the final wash temperature is decrease by 5°C.). In practice, the change in T_(m) can be between 0.5° C. and 1.5° C.per 1% mismatch.

Stringent conditions involve hybridizing at 68° C. in 5×SSC/5×Denhart'ssolution/1.0% SDS, and washing in 0.2×SSC/0.1% SDS at room temperature.Moderately stringent conditions include washing in 3×SSC at 42° C. Theparameters of salt concentration and temperature be varied to achieveoptimal level of identity between the primer and the target nucleicacid. Additional guidance regarding such conditions is readily availablein the art, for example, Sambrook, Fischer and Maniatis, MolecularCloning, a laboratory manual, (2nd ed.), Cold Spring Harbor LaboratoryPress, New York, (1989) and F. M. Ausubel et al eds., Current Protocolsin Molecular Biology, John Wiley and Sons (1994).

This aspect of the invention involves use of a method for reliableextraction of RNA from an FPE specimen and second, determination of thecontent of DPD mRNA in the specimen by using oligonucleotide primersoligionucleotide primer pair DPD3A (DPD3a-51F (SEQ ID NO: 1) andDPD3a-134R (SEQ ID NO: 2)) or oligonucleotides substantially identicalthereto or DPD3B (DPD3b-651F (SEQ ID NO: 7) and DPD3b-736R (SEQ ID NO:8)) or oligonucleotides substantially identical thereto, for carryingout reverse transcriptase polymerase chain reaction. RNA is extractedfrom the FPE cells by any of the methods for mRNA isolation from suchsamples as described in U.S. patent application Ser. No. 09/469,338,filed Dec. 20, 1999, and is hereby incorporated by reference in itsentirety.

The present invention resides in part in the finding that the relativeamount of DPD mRNA is correlated with resistance to the chemotherapeuticagent 5-FU. It has been found herein that tumors expressing high levelsof DPD mRNA are considered likely to be resistant to 5-FU. Conversely,those tumors expressing low amounts of DPD mRNA are likely to besensitive to 5-FU. A patient's relative expression of tumor DPD mRNA isjudged by comparing it to a predetermined threshold expression level ofexpression of DPD.

A predetermined threshold level of DPD mRNA expression, as definedherein, is a level of DPD expression above which it has been found thattumors are likely to be resistant to 5-FU. Expression levels below thisthreshold level are likely to be found in tumors sensitive to 5-FU. Therange of relative expression of DPD, expressed as a ratio of DPD: aninternal control gene, among tumors responding to a 5-FU basedchemotherapeutic regimen responding tumors of less than about 0.6 toabout 2.5, (about a 4.2-fold range). Tumors not responding to a 5-FUbased chemotherapeutic regimen have relative expression of DPD : aninternal control gene ratio of about 0.2 to about 16 (about an 80-foldrange). Tumors generally do not respond to 5-FU treatment if there isDPD expression greater than about 2.0, preferably greater than about2.5.

“Substantially equivalent” as used herein refers to a threshold level ofDPD expression level that is about 2.0 to about 2.5.

The oligonucleotide primers of the invention enable that allow accurateassessment of DPD expression in a fixed paraffin embedded (FPE) tissue.FIG. 1. Additionally, the oligonucleotide primers of the presentinvention have been shown to be accurate for determining DPD expressionlevels in fresh or frozen tissue, i.e. they have high specificity fortheir target RNA. Thus, methods of the invention are not limited to useof paraffin embedded tissue. Oligonucleotide primers disclosed hereinare capable of allowing accurate assessment of DPD gene expression in afixed paraffin embedded tissue, as well as in frozen or fresh tissue.FIG. 2. This is due to the fact that the mRNA derived from FPE samplesis more fragmented relative to that of fresh or frozen tissue and istherefore, more difficult to quantify. Thus, the present inventionprovides oligonucleotide primers that are suitable for use in assayingDPD expression levels in FPE tissue, where previously there existed nosuitable assay. See FIG. 1.

Expression of DPD mRNA is correlated with clinical resistance to5-FU-based chemotherapy. In particular, expression of high levels of DPDmRNA correlate with resistance to 5-FU-based chemotherapies. The presentmethods can be applied to any type of tissue. For example, forexamination of resistance of tumor tissue, it is desirable to examinethe tumor tissue. Preferably, it is desirable to also examine a portionof normal tissue from the patient from which the tumor is obtained.Patients whose normal tissues are resistant to 5-FU-basedchemotherapeutic compounds, but whose tumors are expected to besensitive to such compounds, may then be treated with higher amounts ofthe chemotherapeutic composition.

The methods in this invention are applied over a wide range of tumortypes. This allows for the preparation of individual “tumor expressionprofiles” whereby expression levels of DPD may be determined inindividual patient samples and response to various chemotherapeutics canbe predicted. Most preferably, the methods of the present invention areapplied to bronchalveolar, small bowel or colon tumors. For applicationof some embodiments of the invention to particular tumor types, it ispreferable to confirm the relationship of the measurement to clinicalresistance by compiling a data-set of the correlation of the particularDPD expression parameter measured and clinical resistance to 5-FU-basedchemotherapy.

The methods of the present invention include the step of obtainingsample of cells from a patient's tumor. Solid or lymphoid tumors, orparts thereof are surgically resected from the patient. If it is notpossible to extract RNA from the tissue sample soon after its resection,the sample may then be fixed or frozen. It will then be used to obtainRNA. RNA extracted and isolated from frozen or fresh samples of resectedtissue is extracted by any of the methods typical in the art, forexample, Sambrook, Fischer and Maniatis, Molecular Cloning, a laboratorymanual, (2nd ed.), Cold Spring Harbor Laboratory Press, New York,(1989). Preferably, care is taken to avoid degradation of RNA during theextraction process.

Tissue obtained from the patient may fixed, preferably by formalin(formaldehyde) or gluteraldehyde treatment, for example. Biologicalsamples fixed by alcohol immersion are also contemplated in the presentinvention. Fixed biological samples are often dehydrated and embedded inparaffin or other solid supports known to those of skill in the art.Such solid supports are envisioned to be removable with organicsolvents, allowing for subsequent rehydration of preserved tissue. Fixedand paraffin-embedded (FPE) tissue specimen as described herein refersto storable or archival tissue samples.

RNA is extracted from the FPE cells by any of the methods as describedin U.S. patent application Ser. No. 09/469,338, filed Dec. 20, 1999,which is hereby incorporated by reference in its entirety. Mostpreferably, RNA is extracted from tumor cells from a formalin-fixed andparaffin-embedded tissue specimen.

In an embodiment of the invention, RNA is isolated from an archivalpathological sample or biopsy which is first deparaffinized. Anexemplary deparaffinization method involves washing the paraffinizedsample with an organic solvent, such as xylene. Deparaffinized samplescan be rehydrated with an aqueous solution of a lower alcohol. Suitablelower alcohols, for example include, methanol, ethanol, propanols, andbutanols. Deparaffinized samples may be rehydrated with successivewashes with lower alcoholic solutions of decreasing concentration.Alternatively, the sample is simultaneously deparaffinized andrehydrated.

Once the sample is rehydrated, RNA is extracted from the rehydratedtissue. Deparaffinized samples can be homogenized using mechanical,sonic or other means of homogenization. In one embodiment, rehydratedsamples are homogenized in a solution comprising a chaotropic agent,such as guanidinium thiocyanate (also sold as guanidiniumisothiocyanate).

An “effective concentration of chaotropic agent” is chosen such that atan RNA is purified from a paraffin-embedded sample in an amount ofgreater than about 10 fold that isolated in the absence of a chaotropicagent. Chaotropic agents include: guanidinium compounds, urea,formamide, potassium iodiode, potassium thiocyantate and similarcompounds. The preferred chaotropic agent for the methods of theinvention is a guanidinium compound, such as guanidinium isothiocyanate(also sold as guanidinium thiocyanate) and guanidinium hydrochloride.Many anionic counterions are useful, and one of skill in the art canprepare many guanidinium salts with such appropriate anions. Theeffective concentration of guanidinium solution used in the inventiongenerally has a concentration in the range of about 1 to about 5M with apreferred value of about 4M. If RNA is already in solution, theguanidinium solution may be of higher concentration such that the finalconcentration achieved in the sample is in the range of about 1 to about5M. The guanidinium solution also is preferably buffered to a pH ofabout 3 to about 6, more preferably about 4, with a suitable biochemicalbuffer such as Tris-Cl. The chaotropic solution may also containreducing agents, such as dithiothreitol (DTT) and (β-mercaptoethanol(BME). The chaotropic solution may also contain RNAse inhibitors.

Homogenized samples may be heated to a temperature in the range of about50 to about 100° C. in a chaotropic solution, which contains aneffective amount of a chaotropic agent, such as a guanidinium compound.A preferred chaotropic agent is guanidinium thiocyanate.

RNA is then recovered from the solution by, for example, phenolchloroform extraction, ion exchange chromatography or size-exclusionchromatography. RNA may then be further purified using the techniques ofextraction, electrophoresis, chromatography, precipitation or othersuitable techniques.

The quantification of DPD mRNA from purified total mRNA from fresh,frozen or fixed is preferably carried out using reverse-transcriptasepolymerase chain reaction (RT-PCR) methods common in the art, forexample. Other methods of quantifying of DPD mRNA include for example,the use of molecular beacons and other labeled probes useful inmultiplex PCR. Additionally, the present invention envisages thequantification of DPD mRNA via use of a PCR-free systems employing, forexample fluorescent labeled probes similar to those of the Invader®Assay (Third Wave Technologies, Inc.). Most preferably, quantificationof DPD cDNA and an internal control or house keeping gene (e.g. β-actin)is done using a fluorescence based real-time detection method (ABI PRISM7700 or 7900 Sequence Detection System [TaqMan®], Applied Biosystems,Foster City, Calif.) or similar system as described by Heid et al.,(Genome Res 1996;6:986-994) and Gibson et al.(Genome Res1996;6:995-1001).

As used herein, a “house keeping” gene or “internal control” is meant toinclude any constitutively or globally expressed gene whose presenceenables normalization of mRNA levels. Normalization is a determinationof the overall constitutive level of gene transcription and a controlfor variations in RNA recovery. “House-keeping” genes or “internalcontrols” can include, but are not limited to the cyclophilin gene,β-actin gene, the transferrin receptor gene, GAPDH gene, and the like.Most preferably, the internal control gene is β-actin gene as describedby Eads et al., Cancer Research 1999; 59:2302-2306.

The methods of the invention are applicable to a wide range of tissueand tumor types and so can be used for assessment of treatment in apatient and as a diagnostic or prognostic tool in a range of cancersincluding breast, head and neck, lung, esophageal, colorectal, andothers. Preferably, the present methods are applied to prognosis ofbronchoalveolar, small bowel, and colon cancer.

From the measurement of the amount of DPD mRNA that is expressed in thetumor, the skilled practitioner one can make a prognosis concerningclinical resistance of a tumor to 5-FU-based chemotherapy. 5-FU-basedchemotherapy comprises administration of 5-FU, its derivatives, alone orwith other chemotherapeutics or with a DPD inhibitor such as uracil,5-ethynyluracil, bromovinyluracil, thymine, benzyloxybenzyluracil (BBU)or 5-chloro-2,4-dihydroxypyridine. Furthermore, it has been found thatco-administration of a 5′-deoxy-cytidine derivative of the formula (I)with 5-FU or a derivative thereof results in significantly improveddelivery of 5-FU selectively to tumor tissues as compared with thecombination of 5-FU or its derivative with a DPD inhibitor5-ethynyluracil, and shows significantly improved antitumor activity inhuman cancer xenograft models.

The invention being thus described, practice of the invention isillustrated by the experimental examples provided below. The skilledpractitioner will realize that the materials and methods used in theillustrative examples can be modified in various ways. Suchmodifications are considered to fall within the scope of the presentinvention.

EXAMPLES Example 1 RNA Isolation from FPE Tissue

RNA is extracted from paraffin-embedded tissue by the following generalprocedure.

A. Deparaffinization and Hydration of Sections:

-   -   (1) A portion of an approximately 10 μM section is placed in a        1.5 mL plastic centrifuge tube.    -   (2) 600 μL, of xylene are added and the mixture is shaken        vigorously for about 10 minutes at room temperature (roughly 20        to 25° C.).    -   (3) The sample is centrifuged for about 7 minutes at room        temperature at the maximum speed of the bench top centrifuge        (about 10-20,000×g).    -   (4) Steps 2 and 3 are repeated until the majority of paraffin        has been dissolved. Two or more times are normally required        depending on the amount of paraffin included in the original        sample portion.    -   (5) The xylene solution is removed by vigorously shaking with a        lower alcohol, preferably with 100% ethanol (about 600 μL) for        about 3 minutes.    -   (6) The tube is centrifuged for about 7 minutes as in step (3).        The supernatant is decanted and discarded. The pellet becomes        white.    -   (7) Steps 5 and 6 are repeated with successively more dilute        ethanol solutions: first with about 95% ethanol, then with about        80% and finally with about 70% ethanol.    -   (8) The sample is centrifuged for 7 minutes at room temperature        as in step (3). The supernatant is discarded and the pellet is        allowed to dry at room temperature for about 5 minutes.

B. RNA Isolation with Phenol-Chloroform

-   -   (1) 400 μL guanidine isothiocyanate solution including 0.5%        sarcosine and 8 μL dithiothreitol is added.    -   (2) The sample is then homogenized with a tissue homogenizer        (Ultra-Turrax, IKA-Works, Inc., Wilmington, N.C.) for about 2 to        3 minutes while gradually increasing the speed from low speed        (speed 1) to high speed (speed 5).    -   (3) The sample is then heated at about 95° C. for about 5-20        minutes. It is preferable to pierce the cap of the tube        containing the sample before heating with a fine gauge needle.        Alternatively, the cap may be affixed with a plastic clamp or        with laboratory film.    -   (4) The sample is then extracted with 50 μL 2M sodium acetate at        pH 4.0 and 600 μL of phenol/chloroform/isoamyl alcohol        (10:1.93:0.036), prepared fresh by mixing 18 mL phenol with 3.6        mL of a 1:49 isoamyl alcohol:chloroform solution. The solution        is shaken vigorously for about 10 seconds then cooled on ice for        about 15 minutes.    -   (5) The solution is centrifuged for about 7 minutes at maximum        speed. The upper (aqueous) phase is transferred to a new tube.    -   (6) The RNA is precipitated with about 10 μL glycogen and with        400 μL isopropanol for 30 minutes at −20° C.    -   (7) The RNA is pelleted by centrifugation for about 7 minutes in        a benchtop centrifuge at maximum speed; the supernatant is        decanted and discarded; and the pellet washed with approximately        500 μL of about 70 to 75% ethanol.    -   (8) The sample is centrifuged again for 7 minutes at maximum        speed. The supernatant is decanted and the pellet air dried. The        pellet is then dissolved in an appropriate buffer for further        experiments (e.g. 50 pI. 5 mM Tris chloride, pH 8.0).

Example 2 mRNA Reverse Transcription and PCR

Reverse Transcription: RNA was isolated from microdissected ornon-microdissected formalin fixed paraffin embedded (FPE) tissue asillustrated in Example 1 and as previously described in U.S. applicationSer. No. 09/469,338 filed Dec. 20, 1999, which is hereby incorporated byreference in its entirety. After precipitation with ethanol andcentrifugation, the RNA pellet was dissolved in 50 ul of 5 mM Tris/Cl atpH 8.0. The resulting RNA was reverse transcribed with random hexamersand M-MLV from Life Technologies (CAT#28025-02.). The reversetranscription was accomplished by mixing 25 μl of the RNA solution with25.5 μl of “reverse transcription mix” (see below). The reaction wasplaced in a thermocycler for 8 min at 26° C. (for binding the randomhexamers to RNA), 45 min at 42° C. (for the M-MLV reverse transcriptionenzymatic reaction) and 5 min at 95° C. (for heat inactivation ofDNAse).

“Reverse transcription mix” consisted of 10 ul 5× buffer (250 mMTris-HCl, pH 8.3, 375 mM KCl, 15 mM MgCl2), 0.5 ul random hexamers (50O.D. dissolved in 550 ul of 10 mM Tris-HCl pH 7.5) 5 ul 10 mM dNTPs(dATP, dGTP, dCTP and dTTP), 5 ul 0.1 M DTT, 1.25 ul BSA (3 mg/ml in 10mM Tris-HCL, pH 7.5), 1.25 ul RNA Guard 24,800U/ml (RNAse inhibitor)(Porcine #27-0816, Amersham Pharmacia) and 2.5 ul MMLV 200U/ul (LifeTech Cat #28025-02).

Final concentrations of reaction components were: 50 mM Tris-HCl, pH8.3, 75 mM KCl, 3 mM MgCl2, 1.0 mM dNTP, 1.0 mM DTT, 0.00375. mg/ml BSA,0.62 U/ul RNA Guard and 10 U/ul MMLV.

PCR Quantification of mRNA Expression: Quantification of DPD cDNA and aninternal control or house keeping gene (i.e. β-actin, as described inEads et al., (Cancer Research 1999; 59:2302-2306) was done using afluorescence based real-time detection method (ABI PRISM 7700 or 7900Sequence Detection System [TaqMan®], Applied Biosystems, Foster City,Calif.) as described by Heid et al., (Genome Res 1996;6:986-994); Gibsonet al., (Genome Res 1996;6:995-1001). In brief, this method uses a duallabelled fluorogenic oligonucleotide probe (the TaqMan® probe) thatanneals specifically within the template amplicon spanning the forwardand reverse primers. Laser stimulation within the capped wellscontaining the reaction mixture causes emission of a 3′ quencher dye(TAMRA) until the probe is cleaved by the 5′ to 3′ nuclease activity ofthe DNA polymerase during PCR extension, causing release of a 5′reporter dye (6FAM). Production of an amplicon thus causes emission of afluorescent signal that is detected by the TaqMan®'s CCD (charge-coupleddevice) detection camera, and the amount of signal produced at athreshold cycle within the purely exponential phase of the PCR reactionreflecting the starting copy number of the sequence of interest. TaqMan®probe for the oligonucleotide primer pair DPD1 (DPD-70F (SEQ ID NO: 3)and DPD-201R (SEQ ID NO: 4)) is DPD-108Tc (SEQ ID NO:9). TaqMan® probefor the oligonucleotide primer pair DPD2 (DPD2p-1129F (SEQ ID NO: 5) andDPD2p-1208R (SEQ ID NO: 6)) is DPD-2p-1154Tc (SEQ ID NO: 10). TaqMan®probe for the oligonucleotide primer pair DPD3A (DPD3a-51F (SEQ IDNO: 1) and DPD3a-134R (SEQ ID NO: 2)) is DPD3A-71Tc (SEQ ID NO: 11).TaqMan® probe for the oligonucleotide primer pair DPD3B (DPD3b-651F (SEQID NO: 7) and DPD3b-736R (SEQ ID NO: 8)) is DPD3b-685Tc (SEQ ID NO: 12).

Comparison of the starting copy number of the sequence of interest withthe starting copy number of the internal control gene provided arelative gene expression level. TaqMan® analyses yield values that areexpressed as ratios between two absolute measurements (gene ofinterest/internal control gene).

The PCR reaction contained olgionulceotide primers from the pair DPD1(DPD-70F (SEQ ID NO: 3) and DPD-201R (SEQ ID NO: 4)); DPD2 (DPD2p-1129F(SEQ ID NO: 5) and DPD2p-1208R (SEQ ID NO: 6)); DPD3B (DPD3b-651F (SEQID NO: 7), T_(m)=58° C. and DPD3b-736R (SEQ ID NO: 8), T_(m)=60° C.); oroligonucleotide primer pair DPD3A (DPD3a-51F (SEQ ID NO: 1), T_(m)=59°C. and DPD3a-134R (SEQ ID NO: 2), T_(m)=59° C.). Each PCR reactionmixture consisted 0.5 μl of the reverse transcription reactioncontaining the cDNA as well as 600 nM each of both oligonucleotideprimers from only one pair (DPD1, DPD2, DPD3B or DPD3A), 200 nMcorresponding TaqMan® probe (for either DPD1, DPD2, DPD3B or DPD3A), 5 UAmpliTaq Gold Polymerase, 200 μM each dATP, dCTP, dGTP, 400 μM dTTP, 5.5mM MgCl₂, and 1× Taqman Buffer A containing a reference dye, to a finalvolume of less than or equal to 25 μl (all reagents, Applied Biosystems,Foster City, Calif.). Cycling conditions were, 95° C. for 10 min,followed by 45 cycles at 95° C. for 15s and 60° C. for 1 min.

Example 3 DPD Expression in FPE Tumor Samples

The oligonucleotide primer pairs DPD3A (DPD3a-51F (SEQ ID NO: 1) andDPD3a-13R (SEQ ID NO: 2)) and DPD3B (DPD3b-651F (SEQ ID NO: 7) andDPD3b-736R (SEQ ID NO: 8)) allowed robust, reproducible quantitation ofDPD gene expression by RT-PCR using RNA extracted from paraffin-embeddedtissue. FIG. 1. Oligonucleotide primer pair DPD3A (DPD3 a-51F (SEQ IDNO: 1) and DPD3a-13R (SEQ ID NO: 2)) also significantly increased thesensitivity of DPD gene expression analysis by RT-PCR in fresh frozentissue. FIG. 2. RT-PCR was performed using the ABI Prism 7700 SequenceDetection System (Taqman®) as described in Example 2, above.

Thirty cycles were used in the PCR reaction. Each cycle consisted ofdenaturing at 96° C. for 1 min, annealing at 55° C. for 1 min andextending at 72° C. for 2 min. The amplified product usingoligionucleotide primer pair DPD3A (DPD3a-51F (SEQ ID NO: 1) andDPD3a-13R (SEQ ID NO: 2)) was 84 base pairs in length. The amplifiedproduct corresponded to region of DPD cDNA spanning a portion of the 5′untranslated region (UTR) and running into Exon 1. The amplified productusing oligionucleotide primer pair DPD3B (DPD3b-651F (SEQ ID NO: 7) andDPD3b-736R (SEQ ID NO: 8)) is 86 base pairs in length. The amplifiedproduct corresponded to amplifies a region of DPD cDNA corresponding toExon 6.

Oligonucleotide primer pairs DPD3A (DPD3a-51 F (SEQ ID NO: 1) andDPD3a-13R (SEQ ID NO: 2)) and DPD3B (DPD3b-651F (SEQ ID NO: 7), andDPD3b-736R (SEQ ID NO: 8)) were compared to other existing primer setsfor their ability to amplify DPD mRNA derived from 10 different FPEtissue samples. Samples #1-5, and #8-10 were derived from colon, #6 frombronchoalveolar and #7 from small bowel tumor biopsies. Otheroligonucleic acid primer pairs used were DPD1 (DPD-70F (SEQ ID NO: 3)and DPD-201R (SEQ ID NO: 4)) and DPD2 (DPD2p-1129F (SEQ ID NO: 5) andDPD2p-1208R (SEQ ID NO: 6)).

The oligonucleotide primer pair DPD3A (DPD3a-51F (SEQ ID NO: 1) andDPD3a-134R (SEQ ID NO: 2)) was most effective in accurately ascertainingDPD levels in various samples. Oligionucleotide primer pair DPD3B(DPD3b-651F (SEQ ID NO: 7) and DPD3b-736R (SEQ ID NO: 8)) was alsoeffective, yet did not provide as strong a signal. Results illustratedin FIG. 1.

1. An isolated and purified oligonucleotide consisting of the sequenceof SEQ ID NO: 1; wherein said isolated and purified oligonucleotide iscapable of amplifying a portion of the 5′ untranslated region and Exon 1of a Dihydropyrimidine Dehydrogenase (DPD) mRNA isolated from fixed andparaffin embedded (FPE) tumor tissue when used with SEQ ID NO:
 2. 2. Anisolated and purified oligonucleotide consisting of the sequence of SEQID NO: 2; wherein said isolated and purified oligonucleotide is capableof amplifying a portion of the 5′ untranslated region and Exon 1 of aDihydropyrimidine Dehydrogenase (DPD) mRNA isolated from fixed andparaffin embedded (FPE) tumor tissue when used with SEQ ID NO:
 1. 3. Anisolated and purified oligonucleotide consisting of the sequence of SEQID NO: 7; wherein said isolated and purified oligonucleotide is capableof amplifying a portion of Exon 6 of a Dihydropyrimidine Dehydrogenase(DPD) mRNA isolated from fixed and paraffin embedded (FPE) tumor tissuewhen used with SEQ ID NO:
 8. 4. An isolated and purified oligonucleotideconsisting of the sequence of SEQ ID NO: 8; wherein said isolated andpurified oligonucleotide is capable of amplifying a portion of Exon 6 ofa Dihydropyrimidine Dehydrogenase (DPD) mRNA isolated from fixed andparaffin embedded (FPE) tumor tissue when used with SEQ ID NO:
 7. 5. Akit for detecting expression of a Dihydropyrimidine Dehydrogenase (DPD)gene in a tissue comprising at least two oligonucleotide primers, saidtwo oligonucleotide primers consisting of SEQ ID NO: 1; capable ofamplifying a portion of a Dihydropyrimidine Dehydrogenase (DPD) mRNAisolated from fixed and paraffin embedded (FPE) tumor tissue when usedwith SEQ ID NO:2; and SEQ ID NO: 2; capable of amplifying a portion of aDihydropyrimidine Dehydrogenase (DPD) mRNA isolated from fixed andparaffin embedded (FPE) tumor tissue when used with SEQ ID NO:
 1. 6. Akit for detecting expression of a Dihydropyrimidine Dehydrogenase (DPD)gene in a tissue comprising at least two oligonucleotide primers, saidtwo oligonucleotide primers consisting of SEQ ID NO:7 capable ofamplifying portion of a Dihydropyrimidine Dehydrogenase (DPD) mRNAisolated from fixed and paraffin embedded (FPE) tumor tissue when usedwith SEQ ID NO: 8; and SEQ ID NO: 8; capable of amplifying a portion ofa Dihydropyrimidine Dehydrogenase (DPD) mRNA isolated from fixed andparaffin embedded (FPE) tumor tissue when used with SEQ ID NO: 7.